黄芪甲苷对心肌细胞缺氧/复氧损伤的保护作用及其自噬机制研究

张斌; 马倩; 马富珍; 王静; 刘旭东; 何儒华

doi:10.12182/20201260601

黄芪甲苷对心肌细胞缺氧/复氧损伤的保护作用及其自噬机制研究

Astragaloside Ⅳ’s Therapeutic Effect on Myocardial Infarction via Affecting Autophagy and the Mechanism Study

摘要

摘要:
目的研究黄芪甲苷（AS-Ⅳ）对新生大鼠心肌细胞缺氧/复氧（H/R）损伤的保护作用，并探讨其潜在的作用机制。
方法提取并培养新生大鼠心肌细胞，随机分为6组：对照组，H/R组，H/R+低、中、高浓度AS-Ⅳ（AS-Ⅳ终浓度0.1 μmol/L、1 μmol/L、10 μmol/L）组，和H/R+高浓度AS-Ⅳ（10 μmol/L）+AKT抑制剂（5 μmol/L）组。H/R前分组预处理心肌细胞（对照组和H/R组不进行预处理）48 h后，除对照组外，其余各组心肌细胞建立细胞H/R损伤模型。MTT法检测各组心肌细胞活力，实时荧光定量PCR（RT-qPCR）检测自噬基因LC3-Ⅱ、p62的表达，Western blot 检测LC3-Ⅱ、P62蛋白和磷脂酰肌醇3激酶/丝氨酸苏氨酸蛋白激酶（PI3K/AKT）通路蛋白〔包括AKT、磷酸化（p-）AKT（p-AKT）、p-雷帕霉素（p-mTOR）〕和自噬启动因子未配位的51样激酶1（ULK1）的表达，免疫荧光染色检测心肌细胞自噬体P62的表达。
结果 AS-Ⅳ在本研究低、中、高浓度范围内以浓度依赖的方式改善H/R损伤下心肌细胞活力，并抑制H/R损伤后的心肌细胞自噬基因LC3-Ⅱ和p62 mRNA和蛋白的表达，抑制ULK1蛋白的表达（P<0.05），添加AKT抑制剂后，AS-Ⅳ的以上作用被部分抑制（P<0.05）。AS-Ⅳ对AKT和mTOR蛋白的表达没有影响（P>0.05），但可以促进AKT和mTOR的磷酸化（P<0.05）。免疫荧光染色检测结果显示，高浓度AS-Ⅳ可以逆转H/R损伤诱导的自噬体P62的表达。
结论 AS-Ⅳ对H/R损伤心肌细胞具有保护作用，其作用机制可能是通过激活PI3K/AKT途径中的mTOR信号降低自噬水平，从而预防H/R损伤。

Abstract:
Objective The purpose of this study was to investigate the protective effect of astragaloside Ⅳ (AS-Ⅳ) on neonatal rats’ hypoxic/reoxygenated (H/R) injured myocardial cells and to explore its underlying mechanism.
Methods Cardiac cells were extracted from newborn rats and divided into control, H/R, H/R-low AS-Ⅳ (0.1 μmol/L AS-Ⅳ), H/R-medium AS-Ⅳ (1 μmol/L AS-Ⅳ), H/R-high AS-Ⅳ (10 μmol/L AS-Ⅳ) and H/R-high AS-Ⅳ-AKT (10 μmol/L AS-Ⅳ+5 μmol/L AKT) groups. After 48 h of treatment, the contents of LC3-Ⅱ, p62, AKT, pAKT, rapamycin (mTOR) mammalian targets and uncoordinated 51-like kinase 1 (ULK1) in cardiac myocytes were compared. Immunofluorescence staining was used to detect the expression of P62 in myocardium autophagosome.
Restults AS-Ⅳ improved the proliferative activity of cardio AS-Ⅳ improved the proliferative activity of cardiomyocytes in H/R injury in a dose-dependent manner and inhibited the level of cell autophagy. However, when AKT inhibitors were added, the effect of AS-Ⅳ was partially inhibited (P<0.05). Gene and protein expression showed that AS-Ⅳ had no significant effect on the expression of AKT and mTOR genes (P>0.05), but could significantly promote the phosphorylation of AKT and mTOR (P<0.05). Immunofluorescence staining results showed that high concentrations of the AS - Ⅳ can reverse H/R injury induced the expression of autophagy body P62.
Conclusion AS-Ⅳ showed protection effect on H/R injured myocardial cells. The possible mechanism is by reducing the autophagy level via activating the mTOR signal in the PI3K/AKT pathway, thereby preventing H/R damage in neonatal rat cardiomyocytes.

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